The androgen receptor (AR) is a member of the steroid hormone receptor superfamily. Mutations in AR have been linked to early progression of prostate cancer. However, the mechanism of action of AR in transcriptional regulation and prostate cancer development is unknown. Studies proposed below will address the issue concerning the mechanism by which AR regulates transcription of androgen-responsive genes. The studies are based on a hypothesis that AR exerts its effect by employing coactivators. A robust AR-dependent in vitro transcription system will be established by selecting the strongest AR-dependent promoter and nuclear extracts. The system will be used to biochemically purify and characterize the potential AR-coactivator complex. Several versions of the AR-coactivator complexes will be isolated from different sources, namely, 1) from HeLa or LNCaP cells infected with vaccinia virus containing His6-tagged AR cDNA; 2) from HeLa or LNCaP cells with an integrated copy of FLAG-tagged AR cDNA; 3) from pull-down products using a fusion of GST and the AR ligand binding domain. Purified coactivator complexes will be characterized and tested for its ability to complement high-level activation.